Clyde L. Denis, Ph.D.

Clyde L. Denis, Ph.D.

Professor

Educational Background:

 Ph.D., University of Washington, 1982

Our research focuses on two major areas.  The first involves studying the role of protein aggregation in controlling a number of neurodegenerative diseases, including Alzheimer’s, Parkinson’s, and Huntington’s diseases and Amyotrophic Lateral Sclerosis (ALS, also known as Lou Gehrig’s disease).  The goal of our research for these studies is both to define the exact role of specific aggregates in the etiology of these diseases and to identify therapeutic compounds that interfere with aggregate formation and thereby ameliorate cellular cytotoxicity.  The second focus of our lab is to elucidate the mechanisms that regulate translational initiation, the most important target by which protein synthesis is controlled. 

The neurodegenerative diseases that we study result from aggregation of specific misfolded proteins: for example, the huntingtin (Htt) protein in humans upon acquiring a protein sequence of more than 37 glutamines forms a variety of aggregates that cause cell lethality, memory and neuromuscular defects, and certain death.  Using yeast as a model system for human neurodegenerative diseases, we have been the first group to specifically define uniquely sized Htt aggregates.  Moreover, we have shown that changes in Htt aggregation pattern (from extremely large soluble aggregates, about 5 to 20 MDa, to medium sized soluble aggregates, 1 to 5 MDa, or from insoluble aggregates, up to 200 MDa, to soluble aggregates) are correlated with differences in cell death.  In addition, defects in chaperones (proteins that bind misfolded proteins) have been found to display dramatic effects on Htt aggregation patterns. 

We are currently defining how the sizes and types of Htt aggregates and the protein composition within each of these complexes changes in response to cell growth, chaperones, and the presence of other specific proteins that can become misincorporated into Htt aggregates.  The ability of specific therapeutic compounds to change Htt aggregation phenomenon is being tested so as to provide rapid and reliable means to identify possible factors that could reduce or eliminate Huntington’s disease in humans.

We have also embarked on studying the alpha-synuclein protein that is the primary misfolded protein leading to Parkinson’s disease in humans.  Our studies with alpha-synuclein have identified unique soluble aggregates and have defined several of the key chaperone proteins that are found in these aggregates.  Similar studies with Alzheimer’s disease and ALS aggregates are planned.

In terms of our second focus, protein translation initiates through multiple steps.  The mRNA forms a closed-loop structure in which eIF4E, the mRNA cap binding protein, interacts with the bridging protein, eIF4G, which in turn binds the poly(A)-binding protein (PAB1) that is bound to the poly(A) tail of mRNA.  The closed-loop structure therefore links the 5’ end of the mRNA to the 3’ end.  The resultant complex interacts with the 43S complex that consists of the 40S small ribosomal subunit, translation initiation factors eIF2, -3, -5, and -1 and the charged methionine tRNA to form the 48S complex.  This 48S complex then scans for the initiation codon and brings in the 60S large ribosomal subunit to form the 80S ribosome bound to the mRNA for the commencement of protein synthesis. 

Two major projects are currently being undertaken in our laboratory in regards to translation.  The first concerns defining the absolute abundance of all factors in translating ribosomes as they transit the mRNA from initiation to elongation to termination.  Our present studies have identified a dynamic rearrangement of factors in the translational machinery that occurs as the ribosome moves along the mRNA.  These rearrangements have been linked by our research to possible sites by which protein synthetic processes can be controlled. 

The second emphasis of our translation research involves understanding how translational repression occurs.  Translational initiation can be repressed by a number of viruses, disease states, and stress conditions.  We are currently studying how translational repressors affect the structure and function of both the closed-loop structure and the translating complex.  Translating complexes have been uniquely identified by our lab group using the novel technique of analytical ultracentrifugation with fluorescence detection system.  Repressors such as SBP1, STM1, and SCD6 are being characterized as to which factors they bind and how they cause disruption of translation complexes. 

Representative Publications:

Zhang, C., Wang, W., Park, S., Chiang, Y.-C., Xi, W.,  Laue, T.M., and Denis, C.L. (2014) Only a subset of the PAB1-mRNP proteome is present in mRNA translation complexes. Prot. Sci. 23, 1036-1049. 

Zhang, C., Lee, D.J, Chiang, Y.-C., Richardson, R., Park, S.-W., Wang, X., Laue, T.M., and Denis, C.L. (2013) The RRM1 domain of the poly(A)-binding protein from Saccharomyces cerevisiae is critical to control of mRNA deadenylation. Mol. Genet. Genomics. 288, 401-412.

Richardson, R., Denis, C.L., Zhang, C., Nielsen, M.O., Chiang, Y.C., Kierkegaard, M., Wang, X., Lee, D.,J. Andersen, J.,S. and Yao, G. (2012)  Mass spectrometric identification of proteins that interact with specific domains of the poly(A) binding protein. Mol. Genet. Genomics 288, 401-412.

Wang, X., Zhang, C., Chiang, Y.-C., Toomey. S., Power, M.P., Granoff, M.E., Richardson, R., Lee, D.J., Xi, W., Laue, T.M., and Denis, C.L. (2012) Use of the novel technique of analytical ultracentrifugation with fluorescent detection system identifies a 77S monosomal translation complex.  Prot. Sci.  21, 1253-1268.

Anderson, B., May, C.A., and Denis, C.L. (2012) Identification of ebs1lsm6, and nup159 as suppressors of spt10 efects at ADH2 in Saccharomyces cerevisiae suggests post-transcriptional defects affect mRNA synthesis.  Amer. J. Mol. Biol. 2, 276-285.

Cui, Y., Chiang, Y.-C., Viswanathan, P., Lee, D.J., and Denis, C.L. (2012) SPT5 physically interacts with CCR4 and affects mRNA degradation but does not control mRNA deadenylation. Amer. J. Mol. Biol. 2, 11-20.

Lee, D., Ohn, T., Chiang, Y.-C., Liu, Y,  Quigley, G., Yao, G., and Denis, C.L. (2010) PUF3 acceleration of deadenylation in vivo can operate independently of CCR4 activity, possibly involving effects on the PAB1-mRNP structure. J. Mol. Biol. 399, 562-575.

Govindan, M, Meng, X, Denis, C.L., Webb, P., Baxter, J., and Walfish, P.  (2009) Identification of CCR4 and other essential thyroid hormone receptor coactivators by modified yeast synthetic genetic array analysis. Proc. Natl. Acad. Sci. USA. 106, 19854-19859.

Cui, Y., Ramnarain, D.B., Chiang, Y.-C., Ding, L.-H., McMahon, J.S., and Denis, C.L. (2008) Genome wide expression analysis of the CCR4-NOT complex indicates that it consists of three modules with the NOT module controlling SAGA-responsive genes. Mol. Genet. Genomics 279, 323-337.

Yao, G., Chiang, Y.-C., Zhang, C., Lee, D., Laue, T.M., and Denis, C.L. (2007) PAB1 self-association precludes its binding to poly (A), thereby accelerating CCR4 deadenylation in vivo. Mol. Cell. Biol. 27, 6243-6253.

Ohn, T., Chiang, Y.-C., Lee, D.J., Yao, G., Zhang, C., and Denis, C.L. (2007) CAF1 plays an important role in mRNA deadenylation separate from its contact to CCR4. Nucl. Acids Res. 35, 3002-3015.

Traven, A., Hammet, A., Tenis, N., Denis, C.L., and Heierhorst, J. (2005). Ccr4-Not complex mRNA deadenylase activity contributes to DNA damage responses in Saccharomyces cerevisiae. Genetics. 169, 65-75.

Viswanathan, P., Ohn, T., Chiang, Y.-C., Chen, J., and Denis, C.L. (2004) Mouse CAF1 can function as a processive deadenylase/3’-5’ exonuclease in vitro but in yeast the deadenylase function of CAF1 is not required for mRNA poly(A) removal. J. Biol. Chem. 279, 23988-23995.

Clark, L.B., Viswanathan, P., Quigley, G., Chiang, Y.-C., McMahon, J.S., Yao, G., Chen, J., Nelsbach, A., and Denis, C.L. (2004) Systematic mutagenesis of the leucine-rich repeat (LRR) domain of CCR4 reveals specific sites for binding to CAF1 and a separate critical role for the LRR in CCR4 deadenylase activity. J. Biol. Chem. 279, 13616-13623.

Cui, Y. and Denis, C.L. (2003) In vivo evidence that defects in the transcriptional elongation factors RPB2, TFIIS, and SPT5 enhance upstream poly (A) site utilization. Mol. Cell. Biol. 23, 7887-7901.

Denis, C.L. and Chen, J. (2003) The CCR4-NOT complex plays diverse roles in mRNA metabolism. Prog. Nucl. Acids Res. 73, 221-250.

Viswanathan, P., Chen, J., Chiang, Y.-C., and Denis, C.L. (2003) Identification of multiple RNA features that influence CCR4 deadenylation activity. J. Biol. Chem. 278, 14949-14955.

Russell, P., Benson, J.D., and Denis, C.L. (2002) Characterization of mutations in NOT2 indicates that it plays an important role in maintaining the integrity of the CCR4-NOT complex. J. Mol. Biol. 322, 27-39.

Chen, J., Chiang, Y.-C., and Denis, C.L. (2002). CCR4, a 3'-5' poly(A) RNA and ssDNA exonuclease, is the catalytic component of the cytoplasmic deadenylase. EMBO J. 21, 1414-1426.

Chen, J., Chiang, Y.-C., Rappsilber, J., Russell, P., Mann, M., and Denis, C.L. (2001). Purification and characterization of the 1.0MDa CCR4-NOT complex identifies two novel components of the complex. J. Mol. Biol. 314, 683-694.

Denis, C.L., Chiang, Y.-C., Cui, Y., and Chen, J. (2001). Genetic evidence supports a role for the yeast CCR4-NOT complex in transcriptional elongation. Genetics 158, 627-634.

Tucker, M., Valencia-Sanchez, M.A., Staples, R., Chen, J., Denis, C.L., and Parker, R. (2001). The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104, 377-386.

Lui, H.-Y., Chiang, Y.-C., Pan, J., Salvadore, C., Chen, J., Audino, D.C., Badarinarayana, V., Palaniswamy, V., Anderson, B., and Denis, C.L. (2001). Characterization of CAF4 and CAF16 reveal a functional connection between the CCR4-NOT complex and a subset of SRB proteins of the RNA polymerase II holoenzyme. J. Biol. Chem. 276, 7541-7548.

Badarinarayana, V., Chiang, Y.-C., and Denis, C.L. (2000). Functional and physical interactions of the components of CCR4-NOT complex with TBP and its associated factors. Genetics 155, 1045-1054.

Bai, Y., Salvadore, C., Chiang, Y.-C., Collart, M., Liu H.-Y, and Denis, C.L. (1999). The CCR4 and CAF1 proteins of the CCR4-NOT complex are physically and functionally separated from NOT2, NOT4, and NOT5. Mol. Cell. Biol. 19, 6642-6651.

Chang, M., French-Cornay, D., Fan, H.-Y., Klein, H., Denis, C.L., and Jaehning, J.A. (1999). A complex containing RNA polymerase II, Paflp, Cdc73p, Hprlp, and Ccr4p plays a role in protein Kinase C signaling. Mol. Cell. Biol 19, 1056-1067.

Komarnitsky, P.B., Klebenow, E.R., Weil, P.A., and Denis, C.L. (1998) ADR1-mediated transcriptional activational activation requires the presence of an intact TFIID complex. Mol. Cell. Biol 18, 5761-5767.

Komarnitsky, S., Chiang, Y.-C., Luca, F., Chen, J., Toyn, J., Winey, M., Johnston, L.H., and Denis, C.L. (1998). The DBF2 protein kinase binds to and acts through the cell-cycle regulated MOB1 protein. Mol. Cell. Biol. 18, 2100-2107.

Liu, H.-Y., Badarinarayana, V., Audino, D.C., Rappsilber, J., Mann, M., and Denis, C.L. (1998). The NOT proteins are part of the CCR4 transcriptional complex and affect gene expression both positively and negatively. EMBO J. 17, 1097-1106.

Hata, H., Mitsui, H., Liu, H., Bai, Y., Denis, C.L., Shimizu, Y., and Sakai, A. (1998) Dhhlp, a putative RNA helicase, associates with the general transcription factors Pop2p and Ccr4p from Saccharomyces cerevisiae. Genetics 148, 571-579.

Liu, H.-Y, Toyn, J. H., Chiang, Y.-C., Draper, M. P., Johnston, L. H., and Denis, C.L. (1997). DBF2, a cell-cycle regulated protein kinase, is physically and functionally associated with the CCR4 transcriptional regulatory complex. EMBO J. 16, 5289-5298.

 

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